1. Field of the Invention
The present invention relates to a deoxyribonucleic acid (DNA) sensor for measuring DNAs without modification thereof, and to a method of measuring the DNAs by using the sensor.
2. Description of the Related Art
Today, a basic principle of DNA chips which are widely used for analyzing functions of genes and gene expressions is a fluorescence detection method. Accordingly, a laser light source and a complicated optical system are required. As a result, a measurement system is large and high-priced. DNA probes used in this case are required to be labeled by using a fluorescent substance, and moreover, a cleaning operation (bound free separation; BF separation) is needed for removing free fluorescence-labeled DNA probes after binding fluorescence-labeled DNA probes to target DNAs (i.e., hybridization).
Methods of measuring the DNAs without modification thereof and without requiring fluorescent substances have been developed in recent years. Such methods, for instance, include: the quartz crystal microbalance (QCM) method configured to immobilize DNA probes on a surface of a quartz resonator and to measure a resonance frequency of the quartz resonator which changes before and after hybridization with target DNAs; and the surface plasmon resonance (SPR) method configured to measure a state change of a liquid on a surface of a sensor before hybridization of target DNAs with DNA probes, which are immobilized on the surface of the sensor by use of the surface plasmon resonance. The QCM measurement basic principle is reduction in the oscillation frequency (frequency variation) caused by adsorption of a substance to an electrode of the quartz resonator, in which a relationship between the oscillation frequency change and a mass of the adsorbed substance is expressed by the following formula called the Sauerbrey formula:
      Δ    ⁢                  ⁢    F    =            -                        2          ⁢                      F            0            2                                                A            ⁡                          (                                                μ                  Q                                ⁢                                  ρ                  Q                                            )                                            1            /            2                                ⁢          Δ      m      where, Δm denotes an amount of mass change; F0 denotes fundamental oscillation frequency; ΔF denotes an amount of change in fundamental oscillation frequency; A denotes an area of an electrode; μQ denotes shear modulus of quartz; and ρQ denotes density of quartz.
The change in the mass of the quartz resonator on the electrode is proportional to the change in the oscillation frequency. For example, it is confirmed that, when the quartz resonator having a fundamental oscillation frequency F0 (Hz) of 27 MHz is used in the air, a 1 Hz oscillation frequency is reduced by adsorption of a substance in an amount of 0.62 ng to each 1 cm2 of an electrode. In the case of single base extension where a single base is added, assuming that immobilization density of DNAs is 4×1012 molecules/cm2 and that a change in the molecular mass per a single base extension is approximately 300, extension of a single base causes a change in the mass by 2.0×10−9 g/cm2. This value is equivalent to an amount of change in frequency by about 3 Hz. However, the single base extension comes under the influence of a change in the viscosity of a solvent under the condition in which the quartz resonator is actually used in a liquid (Anal. Chim. Acta 175 (1985) 99-105). The influence of the change in the viscosity of the solvent is expressed by the following formula:ΔF=−F02/3(ρnηL/πρQηQ)1/2 where ρL denotes density of solvent; ηL denotes viscosity of solvent; ρQ denotes density of quartz; and ηQ denotes shear modulus of quartz.
This indicates that actual measurement is influenced by a change in the temperature in addition to a pulsating flow in introducing a sample and to a change in solution composition. For example, as for the influence of the temperature change in the case of water, the change in the viscosity is dominant. In this case, the change ratio in the viscosity is 2%/° C., and the change in the frequency is approximately 1000 Hz/° C. This value means that a change in the temperature of 1° C. is equivalent to a change in the mass of 6.0×10−7 g/cm2. For this reason, the QCM method requires a temperature-controlled bath and a liquid pumping-system, which are highly accurate, in order to reduce these influences. As a result, an apparatus therefor is large-scaled and complicated. In the measurement where the temperature is actually controlled, frequency fluctuations range from 16 Hz to 24 Hz approximately, and the minimum limit of detection for the change in the mass ranges from 1.0×10−8 to 1.5×10−8 g/cm2 (Langmuir 9, (1993) 574-576pp., J. Am. Chem. Soc. 120, (1998) 8537-8538). As described above, the QCM method is sensitive to the temperature change and has a difficulty for measurement of the change in the mass of 2.0×10−9 g/cm2 in the case of a single base extension reaction. Similarly, the SPR method is influenced by the temperature change in addition to the pulsating flow in introducing the sample and to the change in the solution composition.
On the other hand, some methods which are paid attention to as small-size and simple methods include the pyrosequencing method and a filed effect transistor (FET) sensor. The pyrosequencing method is configured to hybridize target DNAs with DNA probes, convert pyrophosphate generated in a complementary strand extension reaction to adenosine triphosphate (ATP), cause this ATP to emit light by use of a luciferin-luciferase luminescence system, identify a substrate (deoxyribonucleoside triphosphate) incorporated in the complementary strand extension reaction by detecting this bioluminescence, and then, determine a base sequence sequentially from the adjacent regions of a primer (Anal. Chem. Acta. 175, (1985) 99-105 pp., Japanese Patent Translation Publication No. 2001-506864, and Japanese Patent Publication No. 3510272). The FET sensor is configured to immobilize DNA probes on a gate insulating layer formed on a space between a source and a drain, and to detect, as a change in a current value between the source and the drain, surface potential on an insulating film generated by hybridization of the DNA probes with target DNAs (Japanese Patent Translation Publication No. 2001-511245). In these methods, the detecting method by using the bioluminescence is a promising method as a detecting method capable of detecting the presence or absence of hybridization of the target DNAs with the DNA probes without using a fluorescent substance label or BF separation.
The above-described pyrosequencing method using the bioluminescence method is configured of three enzyme reaction processes including an extension reaction by DNA polymerase, a reaction to convert pyrophosphate generated by the extension reaction to ATP (for example, ATP sulfurylase), and a reaction to cause light emission with a luciferin-luciferase luminescence system by utilizing ATP generated by an ATP conversion enzyme. Accordingly, the respective enzymes need different reagents such as substrates. Moreover, the enzyme reactions used herein have respectively different optimal reaction conditions, and thereby. it is necessary to adjust the reaction conditions in order for the respective enzymes to act as much as possible. In addition, in deoxyribonucleoside triphosphate selected from a group consisting of dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate) and dTTP (deoxythymidine triphosphate) used for the extension reaction, the dATP is a pseudosubstrate (i.e., a luminescent substance) of a luciferin-luciferase reaction and is therefore a noise source. Accordingly, it is necessary to use deoxyadenosine α-thiotriphosphate (dATPαS) as an analog (Japanese Patent Publication No. 3510272). The dATPαS is more expensive than the dATP, while the dATPαS has problems of poor reactivity as a DNA polymerase substrate, and of poor thermostability to cause the decomposition thereof easily. Meanwhile, since the deoxyribonucleoside triphosphate contains the pyrophosphate which is the substrate of this reaction, it is necessary to perform a complicated process to decompose the pyrophosphate by use of an enzyme such as pyrophosphatase. In order to perform processing of numerous samples at the same time, it is necessary to minimize a reaction tank and to perform a two-dimensional array at high density. In this case, there are problems: of deterioration in sensitivity owing to reduction in the amount of light emission with the reaction tank downsizing; and of crosstalk in which light emission is leaked within the reaction tanks with the density in the two-dimensional array increasing.
Meanwhile, in principle, the FET sensor can detect, as a potential change, an increased amount of a phosphate group added by the extension reaction of the DNA probes immobilized on the gate insulating layer formed on a space between the source and the drain. As compared to luminescence detection, the FET sensor has an advantage of requiring fewer reagents (such as enzymes or substrates) used therefor. Nevertheless, in the conventional technique, the DNA probes are immobilized on the gate insulating layer in the following manner: an amino group is introduced by chemically modifying the surface of the gate insulating film by use of aminopropylsilane, polylysine or the like; and the DNA probes each having an end which is chemically modified with the amino group by use of glutaraldehyde or phenylenediisocyanate. Therefore, this technique requires the complicated preprocessing. In recent years, an extended gate FET has been disclosed (Japanese Patent Publication No. 2005-77210), and in the extended gate FET, a gold electrode for DNA probe immobilization is connected to a gate of an insulated gate field effect transistor with a conductive wire. By using the gold electrode in a sensing area which is a DNA immobilization area, the specific binding between gold and a thiol can be applied. Accordingly, by using the specific binding, the DNA probes each including alkanethiol as a linker can be easily immobilized to the gold electrode. However, the DNA probe immobilization form suitable for the extended gate FET sensor and the usage thereof have not yet been studied in detail. In particular, a relation between hybridization efficiency and immobilization density of the DNA probes, the relation having large influence on detection sensitivity, and a method of reducing an influence of a disturbance factor in a solution due to foreign substances and the like have not been clarified. In addition, a suitable array of sensors and the like for simultaneously processing numerous samples have not also been made clear.